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High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors

activity and production of infectious virus (data not shown).

DNA Template
(kb)

Extension Time
(minutes/kb)

Number of Transformantsa

QuikChange kit

QuikChange XL kit

10.1

1

0

32

aReactions were carried out as described in the text and kit manuals. Values represent mean number of transformants from 3 plates.

Small-to-moderately sized plasmid DNA templates can be efficiently mutagenized with either QuikChange kit4. For such targets, the QuikChange XL kit permits the use of reduced reaction times. For example, the 4.5-kb kit control was efficiently replicated with the QuikChange XL kit using 0.5 min/kb extension times rather the 2 minute/kb extension times recommended for the standard QuikChange kit (data not shown). In these experiments, the average mutation frequency for the kit control was 98%, indicating that QuikChange XL kit retains the high mutagenesis efficiency of the QuikChange site-directed mutagenesis method.

Conclusions

We have introduced the QuikChange XL site-directed mutagenesis kit to improve the efficiency of mutagenizing long or difficult-to-mutagenize plasmid DNA templates using this single day mutagenesis method. The QuikChange XL kit features components specifically designed for more efficient DNA replication and bacterial t
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